Methodologies and Reagents for Detecting Fibrinolysis and Hyperfibrinolysis

ABSTRACT

In some embodiments, the invention provides a container adapted for detecting hyperfibrinolysis or fibrinolysis in a blood sample using viscoelastic analysis comprising an interior having a coating comprises an inhibitor of fibrinolysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/270,269 filed May 5, 2014, the entire contents of which ishereby incorporated by reference.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.NIH-T32-GM008315 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND ART

The present invention relates to haemostasis.

Haemostasis is a tightly regulated process which causes bleeding tostop. In the body, circulating blood remains fluid under normalconditions, but forms localized clots when the integrity of the vascularsystem is breeched. Trauma, infection, and inflammation all activate theblood's clotting system, which depends on the interaction of twoseparate systems: enzymatic proteins in a clotting cascade (e.g.,clotting factors such as Factor VII or Factor IX) and activatedplatelets. The two systems work in concert to plug defects in the brokenvessels.

A blood clot (also called a thrombus) that forms during haemostasis ismade of two parts—a platelet plug and a mesh of cross-linked fibrinprotein. The fibrin results from cleavage of fibrinogen into fibrin bythrombin which is activated during the clotting cascade (see FIG. 1). Ablood clot needs to be of sufficient strength to resist dislodgement bycirculating blood or mechanical movement. If a particular clottingfactor is dysfunctional or absent, as in hemophilia, an insufficientamount of fibrin forms. Similarly, massive consumption of clottingfactors in a trauma situation decreases the amount of fibrin formed.Inadequate numbers of platelets resulting from trauma, surgery, orchemotherapy also decrease platelet aggregation, as do geneticdisorders, uremia, or salicylate therapy. Ultimately, reduced fibrinformation or platelet aggregation results in clots of inadequate tensilestrength. This hypocoagulable state makes the patient prone to bleeding.Conversely, endothelial injury, stasis, cancer, genetic diseases, orother hypercoagulable states lead to thrombosis (i.e., blood clot)formation, exemplified in deep-vein thromboses, pulmonary emboli, andarterial occlusions such as stroke and myocardial infarction.

The precursor of plasmin, plasminogen, is an inactive protein that isincorporated into a blood clot. Tissue plasminogen activator (t-PA) andurokinase are able to convert plasminogen to plasmin, thus activating itand allowing fibrinolysis to occur. Fibrinolysis, the process ofbreaking down blood clots, so that they do not become problematic, is anormal biological process. Normally, t-PA is released very slowly intothe blood by the damaged endothelium of blood vessels. As a result,after bleeding is stopped, the clot is broken down as the inactiveplasminogen in the clot is activated to become plasmin, which acts tobreak down the fibrin mesh holding the clot together. The resultingfragments, called fibrin degradation products (FDPs), are then clearedby other enzymes, or by the kidney and liver.

In some situations, hyperfibrinolysis can also occur. This condition, aform of coagulopathy (bleeding disorder) with markedly enhancedfibrinolytic activity, results in increased and sometimes fatalbleeding.

Hyperfibrinolysis can be acquired or can be congenital. Congenitalreasons for hyperfibrinolysis are rare and include deficiency ofalpha-2-antiplasmin (alpha-2-plasmin inhibitor) and deficiency inplasminogen activator inhibitor type 1 (PAI-1). The affected individualsshow a hemophilia-like bleeding phenotype.

Acquired hyperfibrinolysis can occur in patient with liver disease,patients with severe trauma, patients undergoing major surgicalprocedures, and patients with other conditions. Indeed, up to 20% ofseverely injured trauma patients are affected by acquiredhyperfibrinolysis, as are other patients with massive hemorrhage.

Known methods to detect fibrinolysis and hyperfibrinolysis includeindirect immunochemical methods which detect the elevation of biomarkerssuch as D-Dimer (cross-linked fibrin degradation products), fibrinogensplit products (FSP), complexes of plasmin and alpha-2-antiplasmin(PAP). However, the sensitivity and specificity of these methods islimited because elevation of these biomarkers can also occur induced inother conditions. The classical coagulations tests such as PT(prothrombin time), aPPT (activated partial thromboplasin time) orthrombin time are not very sensitive for fibrinolysis andhyperfibrinolysis, and are influenced by numerous other variables.

Thus, there is a need to for methods to rapidly and accurately diagnoseand/or detect fibrinolysis and hyperfibrinolysis.

SUMMARY OF THE EMBODIMENTS

The invention provides methods and reagents to rapidly and accuratelydetect fibrinolysis and hyperfibrinolysis in a blood sample.

Accordingly, in one aspect, the invention provides a method fordetecting fibrinolysis or hyperfibrinolysis in a blood sample. Themethod includes subjecting a first portion of a blood sample comprisingreduced platelet function to viscoelastic analysis in the absence of aninhibitor of fibrinolysis to obtain the coagulation characteristic ofthe first portion at the time point; and subjecting a second portion ofthe blood sample comprising reduced platelet function to viscoelasticanalysis in the presence of an inhibitor of fibrinolysis to obtain acoagulation characteristic of the second portion at a time point;wherein a difference between the coagulation characteristic of the firstportion and the coagulation characteristic of the second portionindicates fibrinolysis or hyperfibrinolysis in the blood sample. In someembodiments, blood sample (e.g., prior to reduction of platelet functionin the blood sample) is taken from a source. In some embodiments, thesource is a blood donation vehicle (e.g., a bag or tube). In someembodiments, the source is a patient is human. In some embodiments, thepatient from whom the blood sample is taken is responsive to theinhibitor of fibrinolysis used in the method.

In some embodiments, the coagulation characteristic is an amplitude ofan output of the viscoelastic analysis. In some embodiments, thecoagulation characteristic a first derivative an amplitude of an outputof the viscoelastic analysis. In some embodiments, the time point is ata time of maximum clot strength of the first portion. In someembodiments, the time point is between about 15 to about 35 minutesafter the viscoelastic analysis is started or is less than 20 minutesafter the viscoelastic analysis is started. In some embodiments, thetime point is obtained is at a time that clot firmness reaches 20 mm inthe blood sample not treated with the inhibitor of fibrinolysis.

In various embodiments, the difference between the coagulationcharacteristic of the first portion and the coagulation characteristicof the second portion that is at least 1% indicates fibrinolysis orhyperfibrinolysis in the blood sample. In further embodiments, thedifference between the coagulation characteristic of the first portionand the coagulation characteristic of the second portion that is atleast 2% indicates fibrinolysis or hyperfibrinolysis in the bloodsample.

In some embodiments, the blood sample comprising reduced plateletfunction comprises an inhibitor of platelet function. In someembodiments, the inhibitor of platelet function is a glycoproteinIIb/IIIa receptor inhibitor, such as abciximab, eptifibatide, ortirofiban. In some embodiments, the inhibitor of platelet function is anadenosine diphosphate (ADP) receptor inhibitor, an adenosine reuptakeinhibitor, or a thromboxane inhibitor. In some embodiments, theinhibitor of platelet function is cytochalasin D. In some embodiments,the inhibitor of platelet function is a combination of differentinhibitors (e.g., a combination of abciximab, eptifibatide, tirofiban,an adenosine diphosphate (ADP) receptor inhibitor, an adenosine reuptakeinhibitor, a thromboxane inhibitor and/or cytochalasin D.

In some embodiments, the blood sample comprising reduced plateletfunction is a platelet-reduced blood sample. For example, theplatelet-reduced blood sample may be obtained by physical removal of theplatelets from the blood sample.

In some embodiments, the inhibitor of fibrinolysis is tranexamic acid.In some embodiments, the inhibitor of fibrinolysis is aminocaproic acid,ε-aminocaproic acid, or aprotinin. In some embodiments, the inhibitor offibrinolysis is a combination of aminocaproic acid, ε-aminocaproic acid,tranexamic acid, and/or aprotinin.

In some embodiments the sample is excited at a resonant frequency andits behavior may be observed by an electromagnetic or light source ascoagulation occurs. In other embodiments the sample's characteristicsmay be observed for changes with a light source without exciting thesample.

In some embodiments, the viscoelastic analysis is performed using ahemostasis analyzer. The hemostasis analyzer may be a TEGthromboelastography analyzer system or in a ROTEM thromboelastometryanalyzer system. In some embodiments, the viscoelastic analysis isperformed using a container containing the sample on an interior of thecontainer and a pin, wherein the pin moves relative to the container. Insome embodiments, the viscoelastic analysis is performed using acontainer containing the sample on an interior of the container and apin, wherein the container moves relative to the pin. In someembodiments the sample is excited at a resonant frequency and itsbehavior may be observed by an electromagnetic or light source ascoagulation occurs. In other embodiments the sample's characteristicsmay be observed for changes with a light source without exciting thesample.

In some embodiments, the inhibitor of fibrinolysis is included in acoating on the interior of the container. In some embodiments, theinhibitor of fibrinolysis is added to the sample in the container. Insome embodiments, the inhibitor of platelet function is included in acoating on the interior of the container. In some embodiments, theinhibitor of platelet function is added to the sample in the container.

In another aspect, the invention provides a method for identifying aninhibitor of fibrinolysis that will achieve a beneficial response in apatient undergoing or likely to undergo fibrinolysis orhyperfibrinolysis, comprising: subjecting a first portion of a bloodsample comprising reduced platelet function to viscoelastic analysis inthe absence of an inhibitor of fibrinolysis to obtain the coagulationcharacteristic of the first portion at the time point; subjecting asecond portion of the blood sample comprising reduced platelet functionto viscoelastic analysis in the presence of first inhibitor offibrinolysis to obtain a coagulation characteristic of the secondportion at a time point; subjecting a third portion of a blood samplecomprising reduced platelet function to viscoelastic analysis in thepresence of first inhibitor of fibrinolysis to obtain a coagulationcharacteristic of the third portion at a time point; and comparing afirst difference between the coagulation characteristic of the firstportion and the coagulation characteristic of the second portion in thepresence of the first inhibitor, and a second difference between thecoagulation characteristic of the first portion and the coagulationcharacteristic of the third portion in the presence of the secondinhibitor, wherein the patient will have beneficial result fromtreatment with the first inhibitor if the first difference is greaterthan the second difference, and the patient will have a beneficialresult from treatment with the second inhibitor if the second inhibitoris greater than the first inhibitor.

In some embodiments, the patient is human.

In some embodiments, the first inhibitor of fibrinolysis is tranexamicacid. In some embodiments, each of the first inhibitor and the secondinhibitor of fibrinolysis is selected from the group consisting ofε-aminocaproic acid, tranexamic acid, aminocaproic acid and aprotinin,wherein the first inhibitor and the second inhibitor are not the same.

In a further aspect, the invention provides a container adapted fordetecting hyperfibrinolysis or fibrinolysis in a blood sample usingviscoelastic analysis comprising an interior having a coating comprisesan inhibitor of fibrinolysis. In some embodiments, the inhibitor offibrinolysis is tranexamic acid. In some embodiments, the inhibitor offibrinolysis is aminocaproic acid, ε-aminocaproic acid, or aprotinin. Insome embodiments, the inhibitor of fibrinolysis is a combination ofaminocaproic acid, ε-aminocaproic acid, tranexamic acid, and/oraprotinin.

In some embodiments, the viscoelastic analysis is performed using a TEGthromboelastography analyzer system or in a ROTEM thromboelastometryanalyzer system. In some embodiments, the inhibitor of fibrinolysis isformulated with sugar in the coating. In some embodiments, the inhibitoris formulated with sodium azide in the coating.

In some embodiments, the coating further comprises an inhibitor ofplatelet function. In some embodiments, the inhibitor of plateletfunction is a glycoprotein IIb/IIIa receptor inhibitor, such asabciximab, eptifibatide, or tirofiban. In some embodiments, theinhibitor of platelet function is an adenosine diphosphate (ADP)receptor inhibitor, adenosine reuptake inhibitor, or a thromboxaneinhibitor. In some embodiments, the inhibitor of platelet function iscytochalasin D. In some embodiments, the inhibitor of platelet functionis a combination of different inhibitors (e.g., a combination ofabciximab, eptifibatide, tirofiban, an adenosine diphosphate (ADP)receptor inhibitor, an adenosine reuptake inhibitor, a thromboxaneinhibitor and/or cytochalasin D.

In some embodiments, the container is located on a cassette or plate,wherein the cassette or plate further comprises a second container.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The foregoing features of embodiments will be more readily understood byreference to the following detailed description, taken with reference tothe accompanying drawings, in which:

FIG. 1 is a schematic diagram showing the clotting cascade that leadseventually to the formation of the fibrin clot made of cross-linkedfibrin. Activation of plasminogen by t-PA produces plasmin whichdegrades the fibrin into fibrin degradation products.

FIG. 2 is schematic diagram showing a TEG tracing from a sample withnormal haemostasis; that is, a normal amount of fibrinolysis and nohyperfibrinolysis. The R (reaction time) is the time of formation of thefibrin strand polymers, K (clot kinetics, measured in minutes) is thespeed at which the clot forms, α is the slope drawn from R to K, and MA(maximum amplitude, measured in mm) is the strength of the clot. TheLY30 is the percent lysis present thirty minutes after the MA.

FIG. 3A is schematic diagram showing a TEMogram tracing. CT indicatesclotting time, CFT indicates clot formation time, alpha is thealpha-angle, lambda-angle is the lysis rate, MCF is the maximum clotfirmness, LI130 is the lysis index 30 minutes after CT, and ML ismaximum lysis.

FIG. 3B is schematic diagram showing another TEMogram tracing.

FIG. 4 is a schematic diagram showing a TEG tracing showing calculationof the LY30 measurement.

FIG. 5 is a schematic diagram showing the differences in TEG tracings ofa blood sample treated with tranexamic acid (a non-limiting fibrinolysisinhibitor) (top line, blue) and a blood sample not treated withtranexamic acid (bottom line, red). In the experiment whose results areshown in this figure, both blood samples were treated with anon-limiting platelet function inhibitor.

FIG. 6 is a schematic diagram showing the differences in TEG tracings ofa blood sample treated with tranexamic acid (a non-limiting fibrinolysisinhibitor) (top line, green) and a blood sample not treated withtranexamic acid (bottom line, blue). In the experiment whose results areshown in this figure, both blood samples were treated with anon-limiting platelet function inhibitor. The LY30 value is superimposedover the TEG tracing of the blood sample not treated with tranexamicacid.

FIG. 7 is a line graph plotting ΔV@MA against increasing concentrationof t-PA.

FIG. 8 is a line graph plotting ΔV@MA against ΔLY30.

FIG. 9 is a line graph plotting LY30 against increasing concentration oft-PA from blood samples of fifteen healthy donors using the citratedkaolin TEG assay.

FIG. 10 is a line graph plotting the ΔLY30 against increasingconcentration oft-PA from blood sample of fifteen healthy donors usingone of the non-limiting methods described herein, where ΔLY30 is thedifference between the LY30 of a functional fibrinogen assay and theLY30 of a functional fibrinogen assay in the presence of an inhibitor offibrinolysis, tranexamic acid.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

In some embodiments, the invention provides methods and reagents (e.g.,cups) for detecting fibrinolysis and hyperfibrinolysis. The inventionstems, in part, from the unexpected discovery that plateletparticipation in the clot can partially mask the onset and extent offibrinolysis. Therefore, reducing platelet function in a blood samplebeing tested for its hemostasis status can speed the detection offibrinolysis and hyperfibrinolysis in the blood sample (and in thepatient from whom the blood sample was taken).

The publications (including patent publications), web sites, companynames, and scientific literature referred to herein establish theknowledge that is available to those with skill in the art and arehereby incorporated by reference in their entirety to the same extent asif each was specifically and individually indicated to be incorporatedby reference. Any conflict between any reference cited herein and thespecific teachings of this specification shall be resolved in favor ofthe latter.

As used herein, the term ‘haemostasis” (or “hemostasis”) is meant aprocess which causes bleeding to stop by the blood coagulating, and alsothe process by which the coagulated blood (or blood clot) dissolves. Theterm includes blood coagulation by formation of a fibrin-containingblood clot, and the breakdown of that clot by activation of plasmin todissolve the fibrin mesh holding the clot together.

Hemostasis is a dynamic, extremely complex process involving manyinteracting factors, which include coagulation and fibrinolyticproteins, activators, inhibitors and cellular elements, such as plateletcytoskeleton, platelet cytoplasmic granules and platelet cell surfaces.As a result, during activation, no factor remains static or works inisolation. Thus, to be complete, it is necessary to measure continuouslyall phases of patient hemostasis as a net product of whole bloodcomponents in a non-isolated, or static fashion. To give an example ofthe consequences of the measuring of an isolated part of hemostasis,assume that a patient developed fibrinolysis, which is caused by theactivation of plasminogen into plasmin, an enzyme that breaks down theclot. In this scenario, a byproduct of this process of fibrinogendegrading product (FDP), which behaves as an anticoagulant. If thepatient is tested only for anticoagulation and is treated accordingly,this patient may remain at risk due to not being treated withantifibrinolytic agents (e.g., treated with tranexamic acid).

The detection of fibrinolysis or hyperfibrinolysis is difficult.Moreover, to be beneficial to a patient who is may need to be treatedwith an antifibrinolytic agent (e.g., during surgery or trauma), thedetection of fibrinolysis or hyperfibrinolysis is preferably very rapid.In some embodiments, the invention provides methods and reagents forrapidly detecting fibrinolysis or hyperfibrinolysis in a blood sample.

Accordingly, in a first aspect, the invention provides method fordetecting fibrinolysis or hyperfibrinolysis in a blood sample,comprising obtaining a blood sample, where the blood sample has areduced platelet function; subjecting a first portion of the bloodsample to viscoelastic analysis in the presence of an inhibitor offibrinolysis to obtain a coagulation characteristic of the first portionat a time point; and subjecting a second portion of the blood sample toviscoelastic analysis in the absence of an inhibitor of fibrinolysis toobtain the coagulation characteristic of the second at the time point;wherein the difference between the coagulation characteristic of thefirst portion and the coagulation characteristic of the second portionindicates fibrinolysis or hyperfibrinolysis in the blood sample.

In some embodiments, the blood sample (e.g., prior to the reduction ofplatelet function in the blood sample) is taken from a source. Thesource can be any source including a donor bag or directly from apatient. In some embodiments, the patient from whom the blood sample istaken is responsive to the inhibitor of fibrinolysis used in the method.

As used herein, the term “fibrinolysis” means the breakdown of a bloodclot due to the conversion of inactive plasminogen in the clot to activeplasmin. During fibrinolysis, active plasmin breaks down the fibrin meshholding the clot together (see FIG. 1).

By “hyperfibrinolysis” is meant a form of coagulopathy (bleedingdisorder) with markedly enhanced fibrinolytic activity, results inincreased and sometimes fatal bleeding. Hyperfibrinolysis can beacquired or can be congenital. Congenital hyperfibrinolysis can be dueto deficiency of alpha-2-antiplasmin (alpha-2-plasmin inhibitor) anddeficiency in plasminogen activator inhibitor type 1 (PAI-1). Acquiredhyperfibrinolysis can occur in patient with liver disease, patients withsevere trauma, patients undergoing major surgical procedures, andpatients with other conditions. Indeed, up to 20% of severely injuredtrauma patients are affected by acquired hyperfibrinolysis, as are otherpatients with massive hemorrhage.

As used herein, by “blood sample” is meant a sample of blood taken, forexample, from a patient. The patient may be a human, but may also be anyother animal (e.g., veterinary animal or exotic animal). Blood is thecirculating tissue of an organism that carries oxygen and nutritivematerials to the tissues and removes carbon dioxide and variousmetabolic products for excretion. Blood consists of a pale yellow orgray yellow fluid, plasma, in which are suspended red blood cells, whiteblood cells, and platelets.

In some embodiments, the blood sample is whole blood. The blood may beuntreated, or may be citrate blood (e.g., whole blood collected into a3.5 mL container containing 3.2% citrate).

By “viscoelastic analysis” is meant any analysis method that measuresthe characteristics of elastic solid (e.g., fibrin solids) and fluids.In other words, viscoelastic analysis allows the study of properties ofa viscous fluid, such as blood or a blood sample. In some embodiments,the viscoelastic analysis is performed under conditions that mimic theconditions in vivo that result in haemostasis. For example, thecondition may include a temperature that mimics a body temperature(e.g., a temperature of 37° C.). The condition may also include clotformation and dissolution at flow rates that mimic those found in bloodvessels.

In some embodiments, viscoelastic analysis of a blood sample may includesubjecting the blood sample to analysis on a hemostasis analyzerinstrument. One non-limiting viscoelastic analysis method is thethromboelastography (“TEG”) assay. Thus in some embodiments, theviscoelastic analysis includes subjecting a blood sample to analysisusing thromboelastography (TEG), which was first described by HelmutHartert in Germany in the 1940's.

Various devices that perform thromboestography, and methods for using itare described in U.S. Pat. Nos. 5,223,227; 6,225,126; 6,537,819;7,182,913; 6,613,573; 6,787,363; 7,179,652; 7,732,213, 8,008,086;7,754,489; 7,939,329; 8,076,144; 6,797,419; 6,890,299; 7,524,670;7,811,792; 20070092405; 20070059840; 8,421,458; US 20120301967; and U.S.Pat. No. 7,261,861, the entire disclosures of each of which are herebyexpressly incorporated herein by reference.

Thromboelastography (TE) monitors the elastic properties of blood as itis induced to clot under a low shear environment resembling sluggishvenous blood flow. The patterns of changes in shear elasticity of thedeveloping clot enable the determination of the kinetics of clotformation, as well as the strength and stability of the formed clot; inshort, the mechanical properties of the developing clot. As describedabove, the kinetics, strength and stability of the clot providesinformation about the ability of the clot to perform “mechanical work,”i.e., resisting the deforming shear stress of the circulating blood. Inessence, the clot is the elementary machine of hemostasis. Haemostasisinstruments that measure haemostasis are able to measure the ability ofthe clot to perform mechanical work throughout its structuraldevelopment. These haemostasis analyzers measure continuously all phasesof patient hemostasis as a net product of whole blood components in anon-isolated, or static fashion from the time of test initiation untilinitial fibrin formation, through clot rate strengthening and ultimatelyclot strength through clot lysis.

In some embodiments, the viscoelastic analysis and/or the haemostasisanalyzer comprises a container which is in contact with the blood.

As used herein, by “container” is meant a rigid surface (e.g., a solidsurface), a portion of which contacts a portion of a blood sample placedinto the container at any point during the viscoelastic analysis. Theportion of the container that contact the portion of blood sample mayalso be referred to as the “interior” of the container. Note that thephase “into the container” does not mean that the container has a bottomsurface which is in contact with the portion of the blood sample.Rather, the container can be a ring-shaped structure, where the insideof the ring is the interior of the container, meaning that the inside ofthe ring is the portion of the ring-shaped container that contacts aportion of the blood sample. A blood sample can flow into the containerand be held there, for example, by vacuum pressure or surface tension.

Still additional types of containers that are included in thisdefinition are those present on plates and cassettes (e.g., amicrofluidic cassette), where the plate or cassette has multiplechannels, reservoirs, tunnels, and rings therein. Each of the contiguouschannels (comprising, for example, a channel, a reservoir, and a ring)is a container, as the term is used herein. Hence, there may be multiplecontainers on one cassette. U.S. Pat. No. 7,261,861 (incorporated hereinby reference) describes such a cassette with multiple channels orcontainers. Any of the surfaces in any of the channels or tunnels of thecassette may be an interior of the container if that surface comes intocontact with any portion of the blood sample, at any time during theviscoelastic analysis.

One non-limiting haemostasis analyzer instrument is described in U.S.Pat. No. 7,261,861; US Patent Publication No. US US20070092405; and USPatent Publication No. US20070059840.

Another non-limiting haemostasis analyzer instrument that performsviscoelastic analysis using thromboelastography is the TEGthromboelastograph hemostasis analyzer system sold commercially byHaemonetics, Corp. (Braintree, Mass.).

Thus, the TEG assay may be performed using the TEG thromboelastographhemostasis analyzer system that measures the mechanical strength of anevolving blood cloth. To run the assay, the blood sample is placed intoa container (e.g., a cup or a cuvette), and a metal pin goes into thecenter of the container. Contact with the interior walls of thecontainer (or addition of a clot activator to the container) initiatesclot formation. The TEG thromboelastograph hemostasis analyzer thenrotates the container in an oscillating fashion, approximately 4.45degrees to 4.75 degrees, every 10 seconds, to imitate sluggish venousflow and activate coagulation. As fibrin and platelet aggregates form,they connect the inside of the container with the metal pin,transferring the energy used to move the container in the pin. A torsionwire connected to the pin measures the strength of the clot over time,with the magnitude of the output directly proportional to the strengthof the clot. As the strength of the clot increases over time, a classicTEG tracing curve develops (See FIG. 2).

The rotational movement of the pin is converted by a transducer to anelectrical signal, which can be monitored by a computer including aprocessor and a control program. The computer is operable on theelectrical signal to create a hemostasis profile corresponding to themeasured clotting process. Additionally, the computer may include avisual display or be coupled to a printer to provide a visualrepresentation of the hemostasis profile. Such a configuration of thecomputer is well within the skills of one having ordinary skill in theart. As shown in FIG. 2, the resulting hemostasis profile (i.e., a TEGtracing curve) is a measure of the time it takes for the first fibrinstrand to be formed, the kinetics of clot formation, the strength of theclot (measured in millimeters (mm) and converted to shear elasticityunits of dyn/cm2) and dissolution of clot. See also Donahue et al., J.Veterinary Emergency and Critical Care: 15(1): 9-16. (March 2005),herein incorporated by reference

The descriptions for several of these measured parameters are asfollows:

R is the time is the period of time of latency from the time that theblood was placed in the TEG 5000 analyzer until the initial fibrinformation. This is typically takes about 30 second to about 10 minutes.For patients in a hypocoagulable state (i.e., a state of decreasedcoagulability of blood), the R number is longer, while in ahypercoagulable state (i.e., a state of increased coagulability ofblood), the R number is shorter.

K value (measured in minutes) is the time from the end of R until theclot reaches 20 mm and this represents the speed of clot formation. ThisK value is about 0 to about 4 minutes (i.e., after the end of R). In ahypocoagulable state, the K number is longer, while in a hypercoagulablestate, the K number is shorter.

α measures the rapidity of fibrin build-up and cross-linking (clotstrengthening) It is angle between the line formed from the split pointtangent to the curve and the horizontal axis. This angle is typicallyabout 47° to 74°. In a hypocoagulable state, the a degree is lower,while in a hypercoagulable state, the α degree is higher.

MA or Maximum Amplitude in mm, is a direct function of the maximumdynamic properties of fibrin and platelet bonding and represents theultimate strength of the fibrin clot. This number is typically fromabout 54 mm to about 72 mm, and the MA occurs typically between about 15to about 35 minutes after the start of the viscoelastic assay. Note thatif the blood sample tested has a reduced platelet function, this MArepresents the strength of the clot based on fibrin only. Decreases inMA may reflect a hypocoagulable state (e.g., with platelet dysfunctionor thrombocytopenia), whereas an increased MA (e.g., coupled withdecreased R) may be suggestive of a hypercoagulable state

LY30 measures the rate of amplitude reduction 30 minutes after MA andrepresents clot retraction, or lysis. The LY30 is thus a percentagedecrease in amplitude 30 minutes after the MA. This number is typically0% to about 8%. In some embodiments, a hypocoagulable state is presentif the LY30 is greater than 7.5%. However, recent findings have shownthat this percentage may be too high to identify a hypocoagulable statein a patient. Accordingly, in some embodiments, an LY30 that is greaterthan 6%, or greater than about 5%, or greater than about 4%, or greaterthan about 3.5%, or greater than about 3% identifies a patient with ahypocoagulable state.

If fibrinolysis occurs, fibrinolysis or dissolution of the clotdecreases the strength of the clot, causing the maximum amplitude (MA)of the TEG tracing to decrease, causing the LY30 percentage to rise (seeFIG. 2).

Another viscoelastic hemostasis assay that can be used is thethromboelastometry (“TEM”) assay. This TEM assay may be performed usingthe ROTEM Thromboelastometry Coagulation Analyzer (TEM InternationalGmbH, Munich, Germany), the use of which is well known (See, e.g.,Sorensen, B., et al., J. Thromb. Haemost., 2003. 1(3): p. 551-8.Ingerslev, J., et al., Haemophilia, 2003. 9(4): p. 348-52.Fenger-Eriksen, C., et al. Br J Anaesth, 2005. 94(3): p. 324-9]. In theROTEM analyzer, the blood sample is placed into a container (also calleda cuvette or cup) and a cylindrical pin is immersed. Between pin and theinterior wall of the container there is a gap of 1 mm which is bridgedby the blood. The pin is rotated by a spring to the right and the left.As long as the blood is liquid (i.e., unclotted), the movement isunrestricted. However, when the blood starts clotting, the clotincreasingly restricts the rotation of the pin with rising clotfirmness. The pin is connected to an optical detector. This kinetic isdetected mechanically and calculated by an integrated computer to thetypical tracing curves (TEMogram) and numerical parameters (see FIGS. 3Aand 3B).

In the ROTEM Thromboelastometry Coagulation Analyzer, the movement ofthe pin can be monitored by a computer including a processor and acontrol program. The computer is operable on the electrical signal tocreate a hemostasis profile corresponding to the measured clottingprocess. Additionally, the computer may include a visual display or becoupled to a printer to provide a visual representation of thehemostasis profile (called a TEMogram. Such a configuration of thecomputer is well within the skills of one having ordinary skill in theart. As shown in FIGS. 3A and 3B, the resulting hemostasis profile(i.e., a TEM tracing curve) is a measure of the time it takes for thefirst fibrin strand to be formed, the kinetics of clot formation, thestrength of the clot (measured in millimeters (mm) and converted toshear elasticity units of dyn/cm2) and dissolution of clot. Thedescriptions for several of these measured parameters are as follows:

CT (clotting time) is the period of time of latency from the time thatthe blood was placed in the ROME analyzer until the clot begins to form.

CFT (Clot formation time): the time from CT until a clot firmness of 20mm point has been reached.

alpha-angle: The alpha angle is the angle of tangent between 2 and thecurve

MA or Maximum Amplitude in mm, is a direct function of the maximumdynamic properties of fibrin and platelet bonding and represents theultimate strength of the fibrin clot. If the blood sample tested has areduced platelet function, this MA is a direct function of the fibrinbonding only.

MCF (Maximum clot firmness): MCF is the greatest vertical amplitude ofthe trace. MCF reflects the absolute strength of the fibrin and plateletclot.

A10 (or A5, A15 or A20 value). This value describes the clot firmness(or amplitude) obtained after 10 (or 5 or 15 or 20) minutes and providea forecast on the expected MCF value at an early stage.

LI30 (Lysis Index after 30 minutes). The LI30 value is the percentage ofremaining clot stability in relation to the MCF value at 30 min afterCT.

ML (Maximum Lysis). The ML parameter describes the percentage of lostclot stability (relative to MCF, in %) viewed at any selected time pointor when the test has been stopped.

A low LI30 value or a high ML value indicates hyperfibrinolysis. Whilein normal blood fibrinolysis activity is quite low, in clinical samplesa more rapid loss of clot stability by hyperfibrinolysis may lead tobleeding complications which can be treated by the administration ofantifibrinolytic drugs.

Thus, parameters of interest in TEG or TEM assays include the maximumstrength of the clot which is a reflection of clot strength. This is theMA value in the TEG assay, and the MCF value in the TEM assay. Thereaction time (R) in TEG (measured in sec) and clotting time (CT) in TEGis the time until there is first evidence of clot; clot kinetics (K,measured in minutes) is a parameter in the TEG test indicating theachievement of clot firmness; and α in TEG or alpha-angle in TEM is anangular measurement from a tangent line drawn to the curve of the TEGtracing or TEM tracing starting from the point of clot reaction timethat is reflective of the kinetics of clot development. (See Trapani, L.M. Thromboelastography: Current Applications, Future Directions”, OpenJournal of Anesthesiology 3(1): Article ID: 27628, 5 pages (2013); andKroll, M. H., “Thromboelastography: Theory and Practice in MeasuringHemostasis,” Clinical Laboratory News: Thromboelastography 36(12),December 2010; instruction manuals for the TEG instrument (availablefrom Haemonetics, Corp.), and the instruction manual for the ROTEMinstrument (available from TEM International GmbH), all of whichdocuments are herein incorporated by reference in their entireties.

In some embodiments, the parameters (and hence the coagulationcharacteristics) are recorded by observation of different excitationlevels of the sample as coagulation occurs. For example, where thecontainer is a microfluidic cassette, or a particular channel in thecassette, the blood sample may be excited at a resonant frequency andits behavior observed by an electromagnetic or light source ascoagulation occurs. In other embodiments the sample's coagulationcharacteristics may be observed for changes with a light source withoutexciting the sample.

Because a single cassette may have multiple containers (e.g., differentchannels in the cassette), the sample in a container contacted with aninhibitor of fibrinolysis is easily directly comparable to a sample in acontainer (e.g., in an adjacent channel in the same microfluidiccassette) that is not contacted with the inhibitor of fibrinolysis.

When no fibrinolysis occurs, the amplitude value at the MA on a TEGtracing and the amplitude value at the MCF on a TEM tracing staysconstant or may decrease slightly due to clot retraction. However, asfibrinolysis occurs (e.g., in a hypocoagulable state), the curve of theTEG tracing and the TEM tracing starts to decay. The resultant loss inpotential area-under-the-curve in the 30 minutes following MaximumAmplitude in the TEG assay is called the LY30 (see FIG. 4). LY30, thepercentage of lysis 30 minutes after the maximum amplitude point(expressed as a percentage of the clot lysed) and clot firmness (G,measured in dynes/cm²), indicates the rate of clot lysis. Thecorresponding value in the TEM assay is the LI30 value (see FIG. 3A)

This LY30 is the usual metric of fibrinolysis. However, this parameterhas some limitations including lack of sensitivity and specificity. Mostimportantly, the LY30 parameter takes at least 30 minutes to obtain. Innormal patients, obtaining the LY30 in at least thirty minutes (becausethe 30 minutes must be added to whatever the time it took the clot toform in the first place) is adequate. However, in some occasions (e.g.,in patients undergoing trauma, hemorrhaging, or during surgery), waitingthe at least 30 minutes to determine if the patient's blood is clottingnormally may be detrimental to the patient's health. For these patientswith a high LY30 who are in a hypocoagulable state, the sooner thepatient can be treated with a fibrinolysis inhibitor (e.g., tranexamicacid or aprotinin).

As used herein, by a “coagulation characteristic” is meant a parameterthat indicates the haemostasis status of the blood sample being tested.For example, the coagulation characteristic may be the amplitude of theoutput of the viscoelastic analysis with respect to a time point. Notethat the time at which the coagulation characteristic is taken need notbe the MA of the sample not treated with the inhibitor of fibrinolysis.The time point can be any time point, so long as the time point is thesame. For example, the time point may be between about 15 to about 35minutes after the start of the viscoelastic analysis assay. The timepoint may also be the moment when the TEG tracing of the inhibitor offibrinolysis-treated sample starts to diverge from the TEG tracing ofthe sample not treated with the inhibitor of fibrinolysis. Thus, thetime point may be as early as about 2 minutes or about 5 minutes afterthe viscoelastic analysis is started.

Other coagulation characteristics measured using a viscoelastic analysisassay can be used in a similar manner to determine if a patient hasfibrinolysis or hyperfibrinolysis. For example, in the TEG assay, any ofR (reaction time), K (time clot firmness is achieved), a (kinetics ofclot development), MA (maximum amplitude), and LY30 can be compared (seeFIGS. 2 and 4). For the TEM assay, any of CT (clotting time), CFT (clotformation time), alpha angle, MCF (maximum clot firmness), A10(amplitude 10 minutes after CT), LI30 (lysis index 30 minutes after CT)and ML (maximum lysis) can be compared (see FIGS. 3A-3B) as well asderivatives of any and all of these parameters can also be used ascoagulation characteristics.

In some embodiments, the derivative of the viscoelastic read-out fromthe blood sample (e.g., a sample that is platelet function-reduced) thatis not treated with an antifibrinolytic agent is compared to thederivative of the viscoelastic read-out from the blood sample that istreated with the antifibrinolytic agent. For historic reasons, theparameters of a TEG or a TEM tracing shows the amplitude (in mm) on they axis and the time on the x axis. The first derivative of a TEG or aTEM tracing is, therefore, velocity (i.e., the slope of the tracingline). The velocity of a tracing from a blood sample not treated with anantifibrinolytic agent can thus be compared to the velocity of theviscoelastic read-out from the blood sample that is treated with theantifibrinolytic agent. This value is called the ΔVA. When there is adifference in the ΔVA, that difference identifies fibrinolysis orhyperfibrinolysis in the sample.

In some embodiments, the ΔVA is obtained at the MA of the sample nottreated with the inhibitor of fibrinolysis. For convenience, thiscoagulation characteristic is referred to as the ΔVA@MA. The ΔVA@MA isdepicted in FIG. 6 as a two-headed arrow between the two tracings at 24minutes.

Note that any of the parameters (e.g., ΔVA, ΔLY30) that are used as thecoagulation characteristic can be obtained by standard methods.Furthermore, the parameters can be obtained using a computer, acalculator, or a computer program or software.

Of course, the second derivative of the velocities can also be obtained,and compared between the sample not treated with the antifribrinolyticagent and the sample treated with the fibrinolytic agent. Indeed, theparameter used as the coagulation characteristic will impact thesensitivity of the methods; however, the method should not be limited toany one particular parameter as a coagulation characteristic. Indeed, ina trauma situation, the routinely skilled physician may simply perform aviscoelastic assay (e.g., a TEG assay) on blood samples (e.g.,platelet-deleted blood samples) from the patient, one without theantifibrinolytic agent and one with the antifibrinolytic agent) in realtime, and once the two tracings begin to diverge (e.g., as early as twominutes after the start of the assay), the physician may choose to treatthe patient with the antifibrinolytic agent at that very moment. Hence,the speed in detecting fibrinolysis or hyperfibrinolysis in a patient isclinically relevant, particularly in the case of trauma patients wherelife and death outcomes can be decided within a matter of minutes.

The methods described herein thus compare a coagulation characteristicin a blood sample treated with the inhibitor of fibrinolysis and thatsame coagulation characteristic in a blood sample not treated with theinhibitor of fibrinolysis. In a patient with normal blood, the selectedcoagulation characteristic in the blood sample treated with theinhibitor of fibrinolysis and that not treated with the inhibitor offibrinolysis will be the same or will be very significantly similar.However, in a patient having fibrinolysis or hyperfibrinolysis, theselected coagulation characteristic in the blood sample treated with theinhibitor of fibrinolysis and that not treated with the inhibitor offibrinolysis will differ. In some embodiments, in a patient withfibrinolysis or hyperfibrinolysis, the difference between thecoagulation characteristic of the blood sample treated with theinhibitor of fibrinolysis and the blood sample not treated with theinhibitor of fibrinolysis will be at least a 1% difference, or at leasta 1.5% difference, or at least a 2% difference, or at least a 2.5%difference, or at least a 3% difference, or at least a 3.5% difference,or at least a 4% difference, or at least a 4.5% difference, or at leasta 5% difference, or at least a 10% difference. As the skilled artisanwill understand, the amount of different will depend, of course, uponthe parameter being used as the coagulation characteristic.

The invention stems, in part, from an attempt to reduce the amount oftime required to detect fibrinolysis and hyperfibrinolysis, by anantifibrinolytic agent (i.e., an inhibitor of fibrinolysis) is added tothe blood sample being tested. Prior to the discovery described in thisdisclosure, the earliest a patient could be identified as havingfibrinolysis or hyperfibrinolysis using a TEG device (e.g., commerciallyavailable from Haemonetics, Corp., Braintree, Mass., USA) was once theLY30 percentage was available. Even under the most rapid circumstances,the LY30 percentage was available no earlier than 43 minutes from thestart of the viscoelastic assay, assuming a reaction time of 4 minutes,a K time of 0 minutes, achieve of the MA at 9 minutes, and 30 minutesfor the LY30 analysis. Typically, the LY30 percentage is not availableuntil at least 50 minutes after the start of the viscoelastic assay. Byskipping the LY30 time constraints, in some embodiments, the methodsdescribed herein can provide results regarding the state of a patient'shemostasis at least 30 minutes earlier than the LY30 percentage could beobtained for that same patient, and in some embodiments even faster than30 minutes earlier than the LY30 percentage could be obtained.

It should be noted that when the “same patient” is referred to, thismeans the same patient at the same time. Hence, a patient who isperfectly healthy individual is not the same patient (for the purposesof this definition) as that same healthy individual who has just beenseverely injured. Obviously, the perfectly healthy uninjured individualmay not have an LY30 percentage at all.

In some embodiments, the time point at which the coagulationcharacteristic is obtained is less than 30 minutes after the start ofthe viscoelastic assay. In some embodiments, the time point at which thecoagulation characteristic is obtained is less than 20 minutes after thestart of the viscoelastic assay, or is less than 15 minutes after thestart of the viscoelastic assay, or is less than 10 minutes after thestart of the viscoelastic assay, or less than 5 minutes after the startof the viscoelastic assay. In some embodiments, the time point at whichthe coagulation characteristic is obtained is at the time of maximumamplitude of the blood sample not treated with the inhibitor offibrinolysis. In some embodiments, the time point at which thecoagulation characteristic is obtained is at the time of maximum clotfirmness of the blood sample not treated with the inhibitor offibrinolysis. In some embodiments, the time point at which thecoagulation characteristic is obtained is at the time that clot firmnessreaches 20 mm in the blood sample not treated with the inhibitor offibrinolysis

In some embodiments, the antifibrinolytic agent is a plasminogeninhibitor. In some embodiments, the plasminogen inhibitor is tranexamicacid (TXA).

In some embodiments, the antifibrinolytic agent is aminocaproic acid(also known as Amicar, ε-aminocaproic acid, or 6-aminohexanoic acid). Insome embodiments, the antifibrinolytic agent is aprotinin.

Inhibitors of fibrinolysis (including those listed above) are well knownand can be used at known concentrations. In various embodiments, theanti-fibrinolytic agent is administered to a blood sample (e.g., a bloodsample with reduced platelet function) at a concentration of betweenabout 2.5 ug/ml to about 250 ug/ml. For those inhibitors of fibrinolysisthat used therapeutically in human patients, dosages are well known andmay be based on individual characteristics of the patient (e.g., stateof overall health, weight, and age).

In some embodiments, the inhibitor of fibrinolysis is added to thecontainer after the blood sample (e.g., having reduced plateletfunction) is added to the container.

In some embodiments, when blood being tested is placed in a container(e.g., a cup or a cuvette), the antifibrinolytic agent is in thecontainer prior to addition of the blood sample.

In some embodiments, the antifibrinolytic agent coats the interior ofthe container such that it is in contact with the blood sample once theblood sample is placed into the container.

During haemostasis, platelets are also involved. Produced bymegakaryocytes in the bone marrow, these small cytoplasmic vesicles,about 1 um in diameter, are full of active biological agents. Just asthe enzymes of the coagulation cascade need to be activated to form afibrin clot, four agents—adenosine diphosphate (ADP), epinephrine,thrombin, and collagen—activate platelets. An adhesive protein calledglycoprotein IIb-IIIa (Gp IIb-IIIa) mediates platelet aggregation. Theprocoagulant factor, fibrinogen, attaches to this receptor, linking theplatelets to each other. The bridging, which is linked by fibrinogen,represents the main source of aggregation. Surgery or trauma exposes theprocoagulant factors to the tissue factor, triggering the coagulationcascade. Besides transforming fibrinogen into fibrin, a polymer thatstrengthens clots, the coagulation cascade produces large amounts ofthrombin, the main activator of platelets.

In some embodiments, the contribution of platelets to a patient's clotformation and strength may be removed or reduced, thereby allowing thedetermination of hyperfibrinolysis or fibrinolysis to be based upon onlythe fibrin content of the clot, and the contribution of fibrinogen.

Thus in some embodiments, the blood sample is a blood sample that hasreduced platelet function. For example, the blood sample may becontacted with a platelet function inhibitor to reduce the function ofthe platelets in the blood sample. The blood sample may also bephysically manipulated (e.g., subjected to centrifugation) to reduce thenumber of platelets in the blood sample by physical removal of theplatelets from the blood sample.

As mentioned above, fibrinogen and platelets both contribute to clotintegrity. In some of the methods described herein, fibrinolysis may bedetected in a blood sample where platelet function has been reduced (forexample by treating the sample with a platelet inhibitor such ascytochalasin D). If fibrinolysis in the platelet function-reduced sampleis prevented with the addition of an anti-fibrinolytic agent (e.g.,tranexamic acid), the fibrinolysis is likely not due to plateletfunction but, rather, to fibrin and other factors in the coagulationcascade. Therefore, the patient from whom the sample was obtained (andwho is prone to develop, or is currently undergoing fibrinolysis orhyperfibrinolysis) will likely respond to treatment with ananti-fibrolytic agent. Thus, in some embodiments, the blood sample beingtested has reduced platelet function as compared to normal whole blood.

Note that by “reduced platelet function” does not mean that the bloodsample does not have any platelet function at all. Rather, the bloodsample with reduced platelet function simply has reduced plateletfunction as opposed to normal whole blood. For example, a blood samplewith reduced platelet function includes a blood sample that has aplatelet function that is at least 25% less, or at least 50% less, or atleast 75% less, or at least 90% less platelet function than whole blood.Platelet function includes, without limitation, the contribution tohemostasis. Reduced platelet function can thus be assessed by areduction in the aggregation of platelets to one another during bloodclotting (e.g., in the presence of Kaolin and calcium).

In some embodiments, the blood sample is physically manipulated toreduce the number of platelets in the blood sample. For example, wholeblood can be centrifuged to remove some or most of the platelets. In onevery simple procedure, 1.5 ul of blood can be centrifuged in a 2.0 mlmicrocentrifuge tube at 1000 rpm for 10 minutes. The platelet-richplasma will float on the top of the blood in the supernatant. Thissupernatant can be removed (e.g., by aspiration) leaving the plateletreduced whole blood at the bottom of the tube. As less than 500 ul ofblood is needed to perform the viscoelastic analyses described below,this is a very rapid method to quickly reduce the number of platelets inthe blood.

In another method, platelet reduced whole blood can be obtained bycontacting whole blood with platelet-specific antibodies attached to asolid surface. The platelets will selectively bind to the solid surface,and the platelet reduced whole blood can be obtained. For example,antibodies that specifically bind to the glycoprotein receptor (which isexpressed on platelets but not on red blood cells) can be coupled tomagnetic beads (e.g., the Dynabeads commercially available from LifeTechnologies, Carlsbad, Calif., USA). Whole blood can be contacted withthe antibody-coated magnetic beads and, after the platelets are allowedto be bound by the antibodies, a magnetic applies. The magnet willattract the beads (and thereby will attract the platelets), and theremaining blood that has a reduced platelet content (and thus a reducedplatelet function) will not be bound to the magnetic and can thus becollected.

In some embodiments, platelet function is reduced by contacting theblood sample with a platelet function inhibitor. One non-limitingplatelet function inhibitor is abciximab (also known as c7E3 Fab).Abciximab is a glycoprotein IIb/IIIa receptor antagonist and inhibitsplatelet aggregation. Additional non-limiting platelet functioninhibitors include adenosine diphosphate (ADP) receptor inhibitors(e.g., clopidogrel, prasugrel, ticagrelor, ticlopidine),phosphodiesterase inhibitors (e.g., cilostazol) glycoprotein IIb/IIIareceptor inhibitors (e.g., abciximab, eptifibatide, and tirofiban),adenosine reuptake inhibitors (e.g., dipyridamole) and thromboxaneinhibitors, including thromboxane synthase inhibitors and thromboxanereceptor antagonists (e.g., tertroban). Any of these platelet functioninhibitors (or combinations thereof) can be used in the methodsdescribed herein.

Platelet function inhibitors (including those listed above andcombinations thereof) are well known and can be used at knownconcentrations to reduce platelet function in whole blood. In variousembodiments, the platelet function inhibitor is administered to a bloodsample (e.g., a whole blood sample) at a concentration of between about2.5 ug/ml to about 250 ug/ml.

In some embodiments of the methods described herein, once a whole bloodsample is collected from the patient, the blood may be treated in such away to reduce platelet function in the sample (e.g., by physicalmanipulation or by contact with a platelet function inhibitor). Forexample, the whole blood can be placed into a single container alreadycontaining an inhibitor of platelet function. Or, an inhibitor ofplatelet function can be added to the container containing whole blood.Or, the whole blood can be platelet depleted (e.g., by physicallyremoving platelets from the blood). Following reduction in plateletfunction, the blood sample can then be separated into the twoviscoelastic assay test groups, with the first test being performed inthe absence of the fibrinolysis agent and the second test beingperformed in the presence of the antifibrinolysis agent.

Of course in some embodiments, the platelet function of the samples isreduced at the same time that the antifibrinolysis agent is added to thesecond test group. Thus, in some embodiments, when blood sample beingtested is placed in a container (e.g., a cup or a cuvette), the plateletfunction inhibitor is in the container prior to addition of the bloodsample. In some embodiments, the platelet function inhibitor coats theinterior of the container such that it is in contact with the bloodsample once the blood sample is placed into the container.

A functional fibrinogen assay using the thromboelastography (TEG)methodology is commercially available from Haemonetics, Corp.(Braintree, Mass., USA). This assay includes a platelet inhibitor andthus removes the contribution of platelets from the measurement offibrinolysis. The use of this functional fibrinogen assay has described(see Harr et al., Shock 39(1): 45-49, 2013).

As described below, to detect fibrinolysis or hyperfibrinolysis at avery early stage, a functional fibrinogen (FF) assay (i.e., a TEG assayremoving the contribution of platelets to the haemostasis process) ismodified by performing the FF assay in the presence or absence of ananti-fibrinolytic agent. Although normal blood will show identical TEGand TEM tracings in both the presence and absence of ananti-fibrinolytic agent, blood from a patient with fibrinolysis orhyperfibrinolysis will show a difference between the tracings in thepresence of the anti-fibrinolytic agent as compared to the tracings inthe absence of an anti-fibrinolytic agent.

In another aspect, the invention provides a method for identifying aninhibitor of fibrinolysis that a patient will be responsive to. Themethod includes subjecting a first portion of the blood samplecomprising reduced platelet function to viscoelastic analysis in theabsence of an inhibitor of fibrinolysis to obtain the coagulationcharacteristic of the first portion at the time point; subjecting asecond portion of a blood sample comprising reduced platelet function toviscoelastic analysis in the presence of first inhibitor of fibrinolysisto obtain a coagulation characteristic of the second portion at a timepoint; subjecting a third portion of a blood sample comprising reducedplatelet function to viscoelastic analysis in the presence of firstinhibitor of fibrinolysis to obtain a coagulation characteristic of thethird portion at a time point; and comparing a first difference betweenthe coagulation characteristic of the first portion and the coagulationcharacteristic of the second portion in the presence of the firstinhibitor, and a second difference between the coagulationcharacteristic of the first portion and the coagulation characteristicof the third portion in the presence of the second inhibitor, whereinthe patient will have beneficial result from treatment with the firstinhibitor if the first difference is greater than the second difference,and the patient will have a beneficial result from treatment with thesecond inhibitor if the second inhibitor is greater than the firstinhibitor.

Of course, the method can include a third inhibitor of fibrinolysis,etc. In some embodiments, each of the first inhibitor and the secondinhibitor of fibrinolysis is selected from the group consisting of(ε-aminocaproic acid), tranexamic acid, and aprotinin, wherein the firstinhibitor and the second inhibitor are not the same. Inhibitors offibrinolysis can be used, for example, within a range of between about2.5 ug/ml to about 250 ug/ml.

In another aspect, the invention provides a container adapted fordetecting the hemostasis status of a blood sample using viscoelasticanalysis comprising an interior having a coating comprising an inhibitorof platelet function. The platelet function inhibitor may be aglycoprotein IIb/IIIa receptor inhibitor (e.g., abciximab, eptifibatide,or tirofiban), or may be an adenosine diphosphate (ADP) receptorinhibitor, adenosine reuptake inhibitor, or a thromboxane inhibitor, ormay be cytochalasin D. In some embodiments, the inhibitor of plateletfunction is a combination of different inhibitors (e.g., a combinationof abciximab, eptifibatide, tirofiban, an adenosine diphosphate (ADP)receptor inhibitor, an adenosine reuptake inhibitor, a thromboxaneinhibitor and/or cytochalasin D.

In another aspect, the invention provides a container adapted fordetecting hyperfibrinolysis or fibrinolysis in a blood sample usingviscoelastic analysis comprising an interior having a coating comprisesan inhibitor of fibrinolysis. In some embodiments, the coating on theinterior of the container further comprises an inhibitor of plateletfunction.

In some embodiments, the inhibitor of fibrinolysis in the coating of thecontainer is tranexamic acid. In some embodiments, the inhibitor offibrinolysis aminocaproic acid (ε-aminocaproic acid), tranexamic acid,or aprotinin. In some embodiments, the inhibitor of fibrinolysis isformulated with sugar and/or sodium azide in the coating.

In some embodiments, the container is used in a viscoelastic analysisperformed using a TEG thromboelastography analyzer system or in a ROTEMthromboelastometry analyzer system.

In some embodiments, the coating of the container further comprises aninhibitor of platelet function. The platelet function inhibitor may be aglycoprotein IIb/IIIa receptor inhibitor (e.g., abciximab, eptifibatide,or tirofiban), or may be an adenosine diphosphate (ADP) receptorinhibitor, adenosine reuptake inhibitor, or a thromboxane inhibitor, ormay be cytochalasin D.

The following examples are provided which are meant to illustrate butnot limit the invention described herein.

Example 1 A Functional Fibrinogen TEG Assay with Tranexamic Acid (TXA)

The methods of Harr et al., supra, are generally followed.

Briefly, citrated whole blood samples are obtained from trauma patients.Venipuncture is performed with a 21-gauge needle in an antecubital vein,and blood is collected into evacuated containers containing 3.2% citrate(e.g., a 3.5 mL plastic Vacutainers® containing 3.2% citrate).

The Functional Fibrinogen assay is purchased from Haemonetics Corp.(Niles, Ill., USA and Braintree, Mass., USA), and performed on the TEG®5000 device according to manufacturer's instructions.

To perform the Functional Fibrinogen (FF) assay, 0.5 mL of citratedblood is added to the designated FF-vial containing a mixture of tissuefactor (a coagulation activator) and the abciximab (a monoclonalGPIIb/IIIa receptor antagonist; sometimes referred to as the FFreagent), and the blood sample is gently mixed. A 340 uL aliquot istransferred from the FF-vial to a 37° C. TEG cup preloaded with 20 μL0.2 mol/L of CaCl2. The FF-assay measures the coagulation parameters ofa platelet-free clot. A second 340 uL aliquot is transferred from theFF-vial to a 37° C. TEG cup preloaded with 20 μL 0.2 mol/L of CaCl2,where the second TEG cup is coated with TXA according to the methoddescribed in Example 2.

The two portions of the blood sample (i.e., the FF without TXA and theFF plus TXA) are analyzed simultaneously on a TEG 5000 device. If theblood sample is normal, the two tracings will be nearly identical andwill form one line (or two very close lines) when overlaid with oneanother.

However, if the blood sample is taken from a patient who hasfibrinolysis or hyperfibrinolysis, the TXA-treated portion of the bloodsample will provide a TEG tracing that is markedly different than theTEG tracing of the portion of the blood same that is not treated withTXA. FIGS. 5 and 6 show representative results from these studies. Asshown, the TXA-treated portion of the blood sample will have a higheramplitude, faster growing TEG tracing. The divergence of these TEGtracing occurs almost immediately (e.g., at 2 minutes after the assaystarts), and is clearly different at between about 15 to about 35minutes after the assay is started (e.g., about 15 to about 35 minutesafter the pin is inserted into the cup). The difference is markedlyclear at the time of the MA of the TEG tracing of the FF alone (i.e.,the FF without TXA)-treated blood sample.

From the data obtained in FIGS. 5 and 6 (or similar results), a newparameter, namely ΔV@MA has been developed. ΔV@MA is the differential invelocity of the two channels (i.e., blood without TXA and blood in thepresence of TXA) at the MA time of the blood sample not treated withTXA. Thus, ΔV@MA can be calculated by simply subtracting the TEGvelocity of the sample not treated with TXA (the bottom line in FIGS. 6and 7) from the TEG velocity of the sample treated with TXA (the toplines in FIGS. 6 and 7).

To determine if this new parameter, namely ΔV@MA is indicative offibrinolysis, first a standard titration curve was created. As discussedabove, t-PA (tissue plasminogen activator) can convert inactiveplasminogen to active plasmin, which can then break down fibrin and thusinduce fibrinolysis. Whole blood from a healthy person was collectedspiked with increasing amounts oft-PA, and the blood sample dividedequally, with TXA being added to one portion but not to the otherportion. The two portions (i.e., two samples) were subjected to analysison a TEG 5000 device, and the ΔV@MA (subtracting the TEG velocity of thesample not treated with TXA from the sample treated with TXA at the MAof the sample not treated with TXA). FIG. 7 is a line graph showingΔV@MA plotted against the concentration of tPA (at ng/ml), revealing astandard titration curve.

The next question was whether this new parameter, ΔV@MA correlated withLY30 which, as discussed above, is the art-known method for detectingfibrinolysis. To do this, a calculation of the ΔLY30 was performed,again calculating the difference between the LY30 number of aTXA-treated sample and an LY30 number of a sample not treated with TXA.The LY30 of a sample not treated with TXA is straight forward. Forexample, referring to FIG. 6, the LY30 of the sample not treated withTXA is approximately 50% (where the MA is approximately 24 minutes intothe assay, and the LY30 is calculated 30 minutes later. For the TXAtreated sample, the LY30 is very small, being approximately 0.1%(calculating the LY30 based on the amplitude of the TXA-treated sampleat 74 minutes). As discussed above, an LY30 as low as 3% may beclinically relevant. Therefore, a ΔLY30 of as low as 3% may also beclinically relevant. The LY30 of roughly 50% in the untreated sample inthis example represents very severe hyperfibrinolysis.

As shown in FIG. 8, within the clinically relevant range of LY30, theΔV@MA versus ΔLY30 curve is linear. Therefore, the new ΔV@MA parameter,which is calculated at the time of the MA of the sample not treated withTXA, and is, by definition, obtained 30 minutes faster than the ΔLY30parameter, is a valid metric of fibrinolysis, but is available to theclinician much faster than the LY30. Such a reduction in the time neededto detect fibrinolysis and hyperfibrinolysis is critically important forthe outcome of trauma patients.

Note that as discussed above, the ΔV need not be obtained at the MA ofthe sample not treated with TXA. Rather, the ΔV can be obtained at anytime the inhibitor of fibrinolysis-treated sample and the sample nottreated with inhibitor of fibrinolysis start showing diverging TEGtracings.

Example 2 Production of Containers Coated with TXA (Tranexamic Acid)

Preparation of the TXA-Coated TEG Cup.

Prepare 15 mL stock using: 1.282 mLs 11.7% trehalose; 0.03 mLs 10% NaN3;0.9 mLs Cyklokapron (commercial preparation of tranexamic acid, 100mg/mL TXA in H2O); and 12.788 mL deionized H2O.

30 uL of this solution is dispensed into a TEG cup, for a final TXAstrength of 180 ug/cup. The cups are air-dried overnight, and packaged20 cups (and pins) per box, with a desiccant pack. Each box is packagedin a sealed Ziploc bag.

The final matrix for the drug solution (before drying) is 1% trehaloseand 0.02% NaN3. Note that the drying process may be performed in alyophilizer, in which case the drying process may be referred to aslyophilization.

Using this method described in Example 2, the amount of TXA in each cupcan be standardized, and the TXA coated cups can be easily stored forlater use.

Example 3 Production of Containers Coated with Either anAnti-Fibrinolytic Agent and an Inhibitor of Platelet Function or withOnly an Inhibitor of Platelet Function

For the cups with both TXA and the platelet inhibitor, a solutioncontaining sodium azide, trehalose, tranexamic acid (TXA), andcytochalasin D is prepared and used to coat the interior of TEG cups. Asdescribed in Example 2, the solution is applied, and then the cupsallowed to dry. The final concentration of TXA in each cup is 180ug/cup. The final concentration of cytochalasin D in each cup is withina range of between about 2.5 ug/ml to about 250 ug/ml.

For the cups with only platelet inhibitor, a solution containing sodiumazide, trehalose, and cytochalasin D is prepared and used to coat theinterior of TEG cups. The solution is applied to the cups, and then thecups allowed to dry. The final concentration of cytochalasin D in eachcup is within a range of between about 2.5 ug/ml to about 250 ug/ml.

Example 4 A TEG Assay with Tranexamic Acid (TXA)

For these studies, the protocol in Example 1 is followed, but the bloodis not citrated and no activator is added to the blood.

Briefly, whole blood is collected from a patient brought in for surgery.The blood sample taken is immediately divided into two portions of 360uL each and each portion is placed into TEG cups. The first portion isadded to a TEG cup coated a coating comprising the FF reagent (amonoclonal GPIIb/IIIa receptor antagonist) and 200 ug tranexamic acid(TXA). The second portion is added to a TEG cup coated with a coatingcomprising 200 ug TXA.

The two portions of the blood sample (i.e., the FF-no TXA and the FFplus TXA) are analyzed simultaneously on a TEG 5000 device. If the bloodsample is normal, the two tracings will be identical and will form oneline when overlaid with one another.

However, at the moment the two tracings start to diverge (e.g., as earlyas two minutes after the start of the analysis), a small dosage of theanti-fibrinolysis agent used in the TEG assay (in this case, tranexamicacid) is prepared for administration to the patient. The dosage may beincreased or not depending upon how much the two tracings diverge. Forexample, if the two tracings diverge extremely at, e.g., 20 minutesafter the start of the analysis, the patient likely hashyperfibrinolysis. At this point, the dosage of the anti-fibrinolysisagent (e.g., tranexamic acid or aprotinin) may be increased to improvethe chances of a favorable outcome for the patient during and followingthe surgical procedure.

Example 5 Comparison of the Citrated Kaolin (CK) TEG to the MethodsDescribed Herein

The method described herein, comparing a coagulation characteristic ofthe patient's blood sample that is not treated with an antifibrinolyticagent to that coagulation characteristic of the patient's blood samplethat is treated with an antifibrinolytic agent is a superior and moresensitive test than the citrated kaolin test.

To prove this concept, blood was taken from 15 donors and separated intotwo groups—the Citrated Kaolin group and the FF (Functional Fibrinogen)group.

For the Citrated Kaolin group, citrated blood from each of the donors(e.g., whole blood collected into a tube containing sodium citrate(thereby creating a ratio blood to citrate of approximately 9:1) iscollected. For eight different tPA concentrations, 0.5 ml of the bloodwas gently mixed with Kaolin (a coagulation activator) and increasingconcentrations oft-PA to induce fibrinolysis. Aliquots were transferredto 37° C. TEG cups, and the samples were analyzed using the TEG 5000device, and the LY30 calculated for each of the 15 donors at each of thetPA concentrations. The results plotting the LY30 against the t-PAconcentration are shown in FIG. 9. As shown by the vertical line in FIG.9, the lysis signal does not rise out of the baseline noise until a t-PAconcentration of between about 75 ng/ml and 100 ng/ml is reached.

For the Functional Fibrinogen (FF) group, citrated blood is collectedfrom each of the donors. For eight different t-PA concentrations, 0.5 mlof the blood was gently mixed with increasing concentrations oft-PA andthe FF reagent (a GPIIb/IIIa receptor antagonist), and the blood sampleswere gently mixed. An aliquot was transferred to a 37° C. TEG cuppreloaded with CaCl2. A second aliquot was transferred to a 37° C. TEGcup preloaded with CaCl2, where the second TEG cup is coated with TXA.

The two TEG cups for each of the fifteen donors at the eight differentt-PA concentrations were analyzed on a TEG 5000 device. From thesetracings, a calculation of the ΔLY30 was performed for each donor. Asdescribed above, the ΔLY30 is simply the difference between LY30 of thesample not treated with TXA (calculated using the amplitudes at the MAtime point and 30 minutes from the MA time point of the untreatedsample) and the LY30 of the TXA-treated sample (calculated using theamplitudes at the MA time point and 30 minutes from the MA time point ofthe TXA-treated sample).

The results plotting the ΔLY30 against the t-PA concentration are shownin FIG. 10. As shown by the vertical line in FIG. 10, the lysis signaloccurs between about 25 and 50 ng/ml t-PA. Thus, the detection thresholdof the method described herein is over 2 times higher than that of thecitrated Kaolin test (compare FIG. 10 to FIG. 9).

Based on these studies, the following sensitivity and specificityparameters were achieved:

Sensitivity Parameters:

-   -   Detection threshold [tPA]: 25-50 ng/mL for ΔLY30; 75-100 ng/mL        for CK-LY30    -   Analytical sensitivity (slope from 75-100 ng/mL): 19.67 for        ΔLY30; 8.01 for CK-LY30    -   RSD of the slope (n=15): 0.57 for ΔLY30; 0.77 for CK-LY30    -   Linear Correlation (R² from 0-200 ng/ml): 0.87 for ΔLY30; 0.72        CK-LY30

Specificity Parameters:

-   -   Baseline signal (mean level below detection threshold): −0.12        for ΔLY30; 7.38 CK-LY30    -   Baseline noise (SD in baseline signal): 0.95 ΔLY30; 4.18 CK-LY30

The embodiments of the invention described above are intended to bemerely exemplary; numerous variations and modifications will be apparentto those skilled in the art. All such variations and modifications areintended to be within the scope of the present invention as defined inany appended claims.

What is claimed is:
 1. A container adapted for detectinghyperfibrinolysis or fibrinolysis in a blood sample using viscoelasticanalysis comprising an interior having a coating comprises an inhibitorof fibrinolysis.
 2. The container of claim 1, wherein the inhibitor offibrinolysis is tranexamic acid.
 3. The container of claim 1, whereinthe inhibitor of fibrinolysis is selected from the group consisting ofan aminocaproic acid (ε-aminocaproic acid), and aprotinin.
 4. Thecontainer of claim 1, wherein the viscoelastic analysis is performedusing a TEG thromboelastography analyzer system or in a ROTEMthromboelastometry analyzer system.
 5. The container of claim 1, whereinthe inhibitor of fibrinolysis is formulated with sugar in the coating.6. The container of claim 1, wherein the inhibitor is formulated withsodium azide in the coating.
 7. The container of claim 1, wherein thecoating further comprises an inhibitor of platelet function.
 8. Thecontainer of claim 7, wherein the inhibitor of platelet function is aglycoprotein IIb/IIIa receptor inhibitor.
 9. The container of claim 8,wherein the glycoprotein IIb/IIIa receptor inhibitor is selected fromthe group consisting of abciximab, eptifibatide, and tirofiban.
 10. Thecontainer of claim 7, wherein the inhibitor of platelet function is anadenosine diphosphate (ADP) receptor inhibitor, adenosine reuptakeinhibitor, or a thromboxane inhibitor.
 11. The container of claim 7,wherein the inhibitor of platelet function is cytochalasin D.
 12. Thecontainer of claim 1, wherein the container is located on a cassette,wherein the cassette further comprises a second container.